abstract book - Clostridia

STRUCTURAL AND GENETIC CHARACTERISATION OF
FLAGELLAR GLYCOSYLATION IN CLOSTRIDIUM BOTULINUM
Susan M. Logan 1 , Catherine J. Paul 1,2 , Evgeny Vinogradov, David J.
McNally, James A. Mullen 1 , David R. McMullin 1 , J.R. Brisson, John W.
Austin 2 , John F. Kelly 1 , and Susan M. Twine 1 .
1 National Research Council Institute for Biological Sciences, Ottawa,
Ontario.
2 Botulism Reference Laboratory, Health Canada, Ottawa,
Ontario.
Flagellins from Clostridium botulinum were shown to be
posttranslationally modified with novel glycan moieties by top down MS
analysis of purified flagellin protein from strains of unique toxin
serotypes. Detailed analysis of flagellin from two strains of C. botulinum
were shown to be modified in O-linkage by a novel glycan moiety of
mass 417 Da. The structural characterisation of the carbohydrate
moiety was completed utilising both MS and NMR and it was shown to
be a novel legionaminic acid derivative. ETD-MS and ETD-cidMS was
successfully utilised to map the 7 sites of O-linked glycosylation
eliminating the need for chemical derivatisation of tryptic peptides prior
to analysis. Marker ions for novel glycans as well as a unique C
terminal flagellin peptide marker ion were identified in a top down
analysis of the intact protein. These ions have the potential to be
utilised for rapid detection and discrimination of C.botulinum cells and
for botulinum neurotoxin (BoNT) contamination. This is the first report of
glycosylation of Gram positive flagellar proteins with the “sialic acid like”
nonulosonate sugar, legionaminic acid. Bioinformatic analysis of
available C. botulinum genomes identified a flagellar glycosylation
island containing homologs of genes recently identified in
Campylobacter coli which have been shown to be responsible for the
biosynthesis of legionaminic acid derivatives.
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